Classic treatments for cancer have been refined and overall survival rates have improved, but better treatments are needed! T cell-based immunotherapy strategies have shown to be an effective therapeutic modality in animal models, however their translation into the clinic, with some notable exceptions, has been disappointing. We hypothesize that immunotherapy strategies fail because patients with cancer have a compromised immune system blocking an effective immune response against tumor antigens. It is becoming increasingly clear that regulatory T cells (Treg) play a critical role in limiting the development of therapeutic anti-tumor responses in preclinical models;and likely play the same role in cancer patients. Our overall hypothesis is that it is possible to eliminate the tumor-induced Treg (iTreg) that limit anti-tumor immune responses without deleting the natural Treg (nTreg) that prevent auto immune disease. Thus, it is critical to develop strategies that can manipulate regulatory T cells induced by cancer, so that vaccines can induce a strong tumor-specific T cell response. We have made progress in increasing the frequency and therapeutic efficacy of tumor-specific effector T cells generated in reconstituted, lymphopenic mice, exploiting homeostasis-driven proliferation. However, when reconstitution used B16BL6-D5 (D5) tumor-bearing mice (TBM) spleen cells, anti-tumor function was lost. Preliminary studies document, that D5 TBM develop CD4+CD25+ tumor-induced regulatory T cells (iTreg) that inhibit the generation of tumor-specific T cells. Further, depletion of CD25+ Treg cells prior to reconstitution successfully restored priming, and gene expression analysis of iTreg cells identified novel markers that can be exploited to deplete iTreg and recover the ability to generate therapeutic T cells in RLM. Some of these genes, which have not previously been associated with regulatory T cells, are up-regulated by iTreg and have signaling properties. This proposal will characterize expression of these novel molecules, investigate their role in the development and/or function of iTreg and explore their use in innovative strategies to increase the priming and therapeutic efficacy of cancer vaccines. Relevance: Results from this proposal will directly influence the translation and advance the design of T cell-based immunotherapy strategies to find a better treatment for patients with progressive disease in current and future clinical trials adopting the findings of these studies to enhance patient therapy.